Viral vectors and the use of the same for gene therapy

ABSTRACT

The invention relates to viral vectors comprising nucleic acid sequences coding for single chain interleukin-12 (single chain IL-12 or scIL12) and a costimulator protein, and to the use of vectors for gene therapy, especially for the treatment of tumors. The invention further relates to adenoviral vectors containing nucleic acid sequences having a sequence homology of at least 90% in relation to the sequence displayed in FIGS.  19  and  20  (IL-12), in FIG.  21  (4-1BB ligand) and in FIG.  22  (IL-2) and optionally also one of the sequences displayed in FIG.  23 A (B7-1) or  23 B (B7-2).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. §371 ofInternational Application Number PCT/EP03/011252, filed Oct. 10, 2003,the disclosure of which is hereby incorporated by reference in itsentirety, and claims the benefit of German Patent Application Number 10248 141.5, filed Oct. 11, 2002.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing is herein incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to viral vectors comprising nucleic acidsequences coding for single chain interleukin-12 (single chain IL-12 orscIL-12) and a costimulator protein, and to the use of said vectors forgene therapy, especially for the treatment of tumors.

Tumors are still one of the most common causes of death of human beingsliving in industrialized countries. For example, the hepatocellularcarcinoma (HCC) is a tumor with an average survival rate of 6 monthsafter diagnosis of one or several larger tumors (Llovet J. M. et al.,Hepatology, 1999, 29: 62-67). The currently used therapies, comprisingradio frequency ablation, chemotherapy and percutaneous ethanolinjection (PEI) result in some success, when treating smaller tumors,but they are insufficient to fight large tumors.

In the prior art, therefore it was suggested to treat HCC by means ofgene therapy. Gene therapeutic treatments are based on theadministration of a nucleic acid that is usually incorporated into thetumor cell, and has a sequence that destroys the tumor cell. To thisend, a multitude of different strategies has been developed that willeffect a destruction of tumor cells caused by the transferred nucleicacid sequences. An overview of such strategies for the treatment of HCCcan be found in Ruiz et al., (Dig. Dis. 2001, 19:324-332). In thispublication, the nucleic acids currently used in clinical trials for thetreatment of HCC in humans, are categorized into one of the fourfollowing groups, according to the treatment strategy:

(1) Transfer of Tumor Suppressor Genes:

-   -   This strategy is based on the fact that the nucleic acid used        for gene therapy contains a gene, which encodes a gene product        that inhibits growth of the tumor or that induces apoptosis in        the tumor cells. Most clinical trials are based on the transfer        of the p53 gene.        (2) Therapy Using Immune Genes    -   This strategy is based on the fact that the nucleic acids used        for gene therapy comprises sequences encoding gene products that        stimulate the patient's immune system and induce an immune        response directed against the tumor cells. The immune response        itself finally results in the destruction of the tumor. Numerous        cytokines, costimulator molecules and tumor-specific molecules        have been suggested for the use in a therapy using immune genes.        (3) Therapy Using Suicide Genes    -   In this procedure, the nucleic acid used for gene therapy        encodes a gene product, for example an enzyme, which transforms        a non-toxic compound into an agent that is cytotoxic for the        tumor cell.        (4) Transfer of Oncolytic Viruses    -   In this variation of gene therapy, nucleic acid vectors are used        that are based on viral sequences. Vectors with oncolytic viral        sequences have a tumor-specific promoter that regulates the        replication of the virus, thus enabling selective viral growth        in the tumor cells.

During the therapy using immune genes (also called immunotherapy) thatis relevant for the current application, nucleic acids are administeredthat comprise sequences, which activate the immune system and aredirected against the tumor. As a general property, the immune systemdoes not only recognize antigens but also tumor-specific structures ontumor cells. Therefore, the activation of the immune system can resultin the destruction of the tumor caused by components of the immunesystem.

According to prior art, numerous molecules are known that stimulate theimmune system or modulate an immune reaction, in particular thecytokines. It was recognized very early that cytokines also haveanti-tumor activities. For example, it was reported that IL-12 is astimulator of the cellular immunity and that it exhibits stronganti-tumor activity (Brunda et al., J. Exp. Med. 1993, 178: 1223-1230).However, the administration of the recombinant IL-12 protein itself asan anti-tumor-agent failed due to the toxic side-effects of the cytokinewhen used in therapeutically relevant doses (Lotze et al., Ann. N.Y.Acad. Sci., 1997, 795: 440-454; and Cohen J., Science, 1995, 270: 908).

Therefore, it was suggested to introduce a nucleic acid encoding acytokine into the tumor, thus enabling a local activation of the immunesystem. Hock et al. (Proc. Natl. Acad. Sci. USA, 1993, 90: 2774-2778)for example, describe the transfer of the interleukin-2 (IL-2),interleukin-4 (IL-4), interleukin-7 (IL-7), TNF or IFN-γ gene into tumorcell lines and the use of these tumor cell lines for the induction oftumors in animals. All transgenic tumor cell lines generated a rejectionreaction against the tumor cells. Dependent on the cytokine used,different cell types of the immune system of the laboratory animals wereinvolved in this rejection reaction (CD4⁺, CD8⁺, CD3⁺).

Vectors coding for IL-12 were also tested for their suitability inimmunotherapy. IL-12, also known as CMLF (cytotoxic lymphocytematuration factor) or NKSF (natural killer cell stimulatory factor), isa heterodimeric cytokine, which is naturally produced by activatedperipheral B-lymphocytes. The protein consists of two subunits withrelative molecular weights of 40 and 35 kDa, respectively, that arecovalently linked by disulfide bridges. The disulfide bridges areessential for the biological activity. As already indicated by thedifferent names, the protein stimulates the proliferation of activatedhuman lyphoblasts and activates natural killer cells.

Vectors coding for the different subunits of this protein were used forthe treatment of tumors (Barajas et al., Hepatology, 2001, 33: 52-61;Mazzolini et al., Cancer Gene Therapy, 1999, 6: 514-522). Furthermore,these vectors were used in combination with other sequences forimmunotherapy. In particular, they were used in combination withsequences coding for a costimulator protein, contained within the sameor in a different vector, for the treatment of tumors (Gyorffy et al.,J. Immunology, 2001, 166: 6212-6217; Martinet et al., Gene Therapy,2002, 9: 786-792; Martinet et al., Journal of National Cancer Institute,2000, 92: 931-936; Guinn et al., J. Immunology, 1999, 162: 5003-5010;and Emtage et al., J. Immunology, 1998, 160: 2531-2538).

Further, IL-12 has already been expressed as single chain IL-12,yielding good activity, such as a protein comprising the differentsubunits linked together in one fusion protein (Lieschke et al., NatureBiotechnology, 1997, 15: 35-40). In a different therapeutic procedure itwas suggested to remove tumor cells from the patient and treat thesecells in vitro with a plasmid coding for single chain IL-12 or IL-12 anda costimulator (US 2002/0018767). Following this in vitro treatment thetumor cells shall be re-implanted into the patient. This procedure,therefore, comprises several operations on the patient and are-implantation of tumor cells into the patient, which is likely toprevent many patients from undergoing such a treatment.

None of the previously used nucleic acids could prevail in the treatmentof mammals, preferably in the treatment of humans. Although, forexample, the publication by Ruiz et al (loc. cit.) described treatmentprocedures involving a very high dosage of the vector used for therapy(3×10⁹−2.5×10¹³ plaque forming units, PFU, per dose), it was observedthat none or only negative results were obtained in the correspondingclinical trials. However, especially the dose of the nucleic acids is acritical factor in gene therapy, because negative side effects or therelease of the vector from the tumor are expected when doses are usedthat are too high.

SUMMARY OF THE INVENTION

The problem of the present invention, therefore, was to provide vectorsthat can be used for immunotherapy with improved efficiency.

This problem was surprisingly solved by using viral vectors comprisingnucleic acid sequences which code for single chain IL-12 and acostimulator protein, wherein the vector is a viral vector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic overview of vectors Ad-1 to Ad-3.

FIG. 2 Determination of the amount of interleukin in the cell culturesupernatant of McA-RH7777 cells after transfection with Ad-1, Ad-2 andAd-3. Vector amounts that should be used for the animal experiments wereadjusted with reference to identical IL-12 expression. FIG. 2 shows thetime course of the expression over 3 days in the rat hepatoma cellsMcA-RH7777.

-   -   Procedure: McA-RH7777 cells were infected at MOIs of 10 with        Ad-1, Ad-2 or Ad-3. The supernatants were collected at days 0,        1, 2 and 3 after infection. scIL-12 concentrations were        determined by ELISA with an anti-mouse IL-12p70 antibody        (Pharmingen).

FIG. 3 Detection of 4-1BBL in the McA-RH7777 cell cultures. Flow-throughcytometric determination of the 4-1BBL expression. 4-1BBL is expressedby Ad-2 and Ad-3, but not by Ad-1.

-   -   Procedure: McA-RH7777 cells were infected with the adjusted        virus concentrations at MOI 10 with Ad-1, Ad-2 or Ad-3. The        cells were harvested 24 h post-infection and were incubated with        a rat anti-mouse 4-1BBL monoclonal antibody (TKS-1, Pharmingen)        and stained with R-PE-conjugated goat anti-rat Ig polyclonal        antibody (Pharmingen) for detection.

FIG. 4 Expression of IL-2 in the McA-RH7777 cell cultures over 3 days.In molar terms Ad-3 expresses 466-fold more IL-12 than IL-2 (calculatedfor day 3).

-   -   Procedure: McA-RH7777 cells were infected with Ad-3 at MOI 10.        The supernatants were collected at days 0, 1, 2 and 3        post-infection. IL-2 concentrations were determined by ELISA        using an anti-mouse IL-2 antibody (Pharmingen).

FIG. 5 Dose escalation study. Change of the tumor size within 9 daysafter treatment with Ad-3.

-   -   Procedure: The tumor volumes were measured by magnetic resonance        tomography (MRT) within a time interval of 9 days. The reference        value of 100% refers to the tumor size at day 3 after virus        injection (1. MRT) the final size shown here was measured at day        12 after virus administration (2. MRT). The vector Ad-3 (a) was        injected at the indicated doses (i.p.=infectious particles) into        tumors of a size between 7 and 11 mm in diameter (b).

FIG. 6 MRT images of the dose escalation study.

-   -   Procedure: Tumors that were treated with 10⁷ to 10⁹ infectious        virus particles of Ad-3 or with 10⁹ infectious particles of        Ad-GFP (negative control), respectively, were scanned by MRT at        day 3 and day 12 post-injection.

FIG. 7 Schematic illustration of the procedure of the animal experimentsusing Ad-1, Ad-2 and Ad-3.

FIG. 8 MRT images of the tumors before the virus injection, week 0.

FIG. 9 MRT images of the tumors after the virus injection, week 3.

FIG. 10 MRT images of the tumors after the virus injection, week 7.

FIG. 11 Course of the changes of the tumor sizes, calculated from theMRT data.

-   -   Procedure: The total volumes were monitored by MRT: sizes were        determined one day before as well as 3 and 7 weeks after virus        administration. Control group Ad-GFP: 9 animals; immune treated        group: 10 animals each in the groups Ad-1, Ad-2 and Ad-3. In        group Ad-1 only one rat displayed continuous tumor growth. All        animals in the control group died within 7 weeks.

FIG. 12 Long-term survival rate of the experimental animals up to 100days after virus injection.

FIG. 13 Map of the vector pTrident3.

FIG. 14 Map of the vector pShuttle [CMV]IL12[IRES]4-1BBL[IRES] IL2.

FIG. 15 Map of the vector pShuttle [CMV]IL12[IRES]4-1BBL.

FIG. 16 Map of the vector pShuttle [CMV]IL12.

FIG. 17 Map of the vector pAd-3.

FIG. 18 Sequence of the tri-cistronic expression cassette containing themurine cDNA; corresponds to insert Ad-3 in FIG. 1 (SEQ ID NO: 1).

FIG. 19 Coding sequence of the human IL-12 40 kDa (SEQ ID NO: 2).

FIG. 20 Coding sequence of the human IL-12 35 kDa (SEQ ID NO. 3).

FIG. 21 Coding sequence of the human 4-1BBL (SEQ ID NO: 4).

FIG. 22 Coding sequence of the human IL-2 (SEQ ID NO: 5).

FIGS. 23A and B Coding sequence of the human B7-1 (SEQ ID NO: 6) andB7-2 (SEQ ID NO: 7).

FIG. 24 Sequence of shuttle vector for Ad-3 (SEQ ID NO: 8).

FIG. 25 Sequence of shuttle vector for Ad-2 (SEQ ID NO: 9).

FIG. 26 Sequence of shuttle vector for Ad-1 (SEQ ID NO: 10).

FIG. 27 Sequence of plasmid containing the whole DNA coding for Ad-3(SEQ ID NO: 11).

FIG. 28 Illustration of the efficacy within a monitoring period of up toone year. The graph shows the fraction of living rats for each timepoint (survival rate 1=100% of the animals in one group). Therapygroups: Ad-3, 5×10⁶ (n=12), Ad-3, 5×10⁷ (n=10). Control group: 5×10⁸Ad-GFP (n=9).

-   -   In this long-term study two liver tumors were planted in each        animal. 2 weeks later (at day 0 in the Figure) one of the tumors        received a single vector injection. At this time point the tumor        volume was approximately 1 ml. The figure shows that the control        animals (treated with Ad-GFP) died within 47 days after vector        injection. In the group treated with 5×10⁶ i.u. (“infectious        units” or infectious particles) of Ad-3 one animal died after 90        days, and in the group treated with 5×10⁷ i.u. of Ad-3 all        animals survived.    -   Rechallenge: In the group treated with 5×10⁷ i.u. of Ad-3,        tumors were again implanted into the liver of all animals at 92        days (13 weeks) after vector injection. The tumor cell implant        disappeared in all 10 animals without further treatment.

FIG. 29 Illustration of the effects on tumor growth after treatment withAd-1 and Ad-3 in comparison with a control vector (Ad-GFP).

-   -   Here, two different dosage levels of Ad-1 and Ad-3 were        administered and tumor sizes were determined 2 weeks        post-treatment. Animal number per group: n=3. The figure shows        the change of tumor volumes at different vector doses of Ad-1        and Ad-3. 1×10⁶ McA-RH7777 tumor cells were injected into the        right liver lobe and 2 weeks later the vector was injected into        the left tumor. MRT scans were performed one day before and 13        days after vector administration. With 5×10⁶ i.u. of Ad-1 the        mean tumor volume increases significantly, whereas it increases        only minimally with 1×10⁷ i.u. of Ad-1. For Ad-3 this only        minimal increase is already observed at a dosage of 5×10⁶ i.u.,        whereas at 1×10⁷ i.u. a decrease becomes already obvious.        Therefore, Ad-3 proves to be clearly more effective than Ad-1.        The values for 1×10⁷ i.u. of Ad-3 were determined at day 3 and        day 12 after vector injection. The controls (Ad-GFP) massively        increase within the monitoring period of 2 weeks. Extrahepatic        metastases were not included into the calculations in those        animals, where they develop.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, the process according to the invention revealed thatvectors coding for single chain IL-12 and a costimulator protein arenotably suitable for the treatment of tumors in the context of animmunotherapy. By means of immunotherapy not only the primary tumor butalso the metastases are eliminated.

The synergistic effect of the constructs according to the inventionencoding proteins involved in the stimulation of the immune systemallows the use of these nucleic acids in lower doses than suggested inthe prior art. This lower dosage results in less side effects, as forexample in a reduced risk for the patient to develop auto-immunediseases, combined with improved security during clinical use. Theprobability of propagating the virus is lower than that using knownviruses. The low dosage further enables the treatment of larger tumormasses or the simultaneous treatment of several tumors withoutgenerating side effects.

Compared to the use of genes coding for both subunits, the use of thesingle chain IL-12 gene saves space in the vector for the expression ofthe nucleic acids. Thus, this sequence allows the use of relativelysmall vectors, for example adenoviral vectors, which nevertheless do notonly contain the gene for single chain IL-12, but also two or moreadditional foreign genes, which intensify the immunotherapy.

The vectors according to the invention are determined for the treatmentof mammals, in particular for the treatment of humans. Therefore, thefollowing references to certain gene sequences preferably refer to humansequences. However, the gene sequences can also be derived from otherspecies or can be modified within the limits of the specified homologousareas using known technologies (which, in the context of the presentapplication, have been determined using BLAST software) as long as thespecific protein activity (immune stimulating and/or T-cell binding)remains in the area of at least 50%, preferably of at least 70% of thecorresponding activity of the human gene product. The quoted areas ofhomology refer to the area, which in the native gene codes for thebiological activity. Provided that nucleic acids are used, which codefor fusion proteins, the area of homology refers to the fragment codingfor the designated biological activity (IL-12, costimulator).Modifications of the gene sequence are included which result in anenhancement of the activity of the proteins.

In the present invention the term “sequences coding for a costimulatorprotein” is used to refer to sequences which, upon expression in humancells, produce proteins that are present as cell surface proteins andwhich are specifically bound by T-cell receptors. Upon binding to aT-cell the costimulator proteins enhance the immune reaction.Corresponding costimulator proteins are known in the art, for example4-1BB ligand (4-1BBL), B7-1 (also known as CD80) and B7-2.

In an especially preferred embodiment of the invention the vectorcomprises sequences, which code for the costimulator protein 4-1BBligand, particularly for sequences having a sequence homology of atleast 40%, preferably of at least 70%, of at least 80% or of at least90% to the sequence shown in FIG. 21, wherein the protein encoded bythis sequence has the ability to bind T-cells specifically. Furtheractivity tests 4-1BBL are known in the art (Vinay D S, Kwon B S. Semin.Immunol., 1998, 10: 481-9. Review; Kwon et al., Mol Cells., 2000, 10:119-26).

In the present invention a protein is referred to as single chain IL-12if the protein consists of an amino acid sequence comprising bothsubunits of the native IL-12 as a fusion protein. Nucleic acid sequencescoding for single chain IL-12 will usually have a sequence homology ofat least 40%, preferably of at least 70%, of at least 80% or of at least90% in relation to the sequences shown in FIGS. 19 and 20. The sequencesshown represent the IL-12 part of the fusion gene. Sequences linking thesubunits are known to the skilled person and are not considered duringhomology analysis. The single chain IL-12 further has an immunestimulating activity, which is not significantly lower than the activityof the native IL-12 in its heterodimeric form. One of the effects of thehuman IL-12 during the stimulation of the immune system is theinitiation of the release of gamma-interferon. The immune stimulatingactivity of single-chain IL-12 is at least 50%, preferably at least 70%of the corresponding activity of the native IL-12. The activity of theproteins can be compared by means of known in vitro assays (for example,by means of the in vitro tests used to compare the activity of IL-12 asdescribed by Lieschke et al., loc. cit.). In an especially preferredembodiment of the invention, the immune stimulating activity of thesingle chain IL-12 is even higher or significantly higher than thecorresponding activity of the native IL-12.

In another embodiment of the invention, the present invention furthercomprises vectors, which code for further cytokines, for proteins withcytokine activity and/or for costimulator proteins. Proteins withcytokine activity are proteins which exhibit the immune stimulatoryactivity of cytokines, but lack any structural relation to cytokinesCorresponding cytokine agonists, for example agonistic cytokine receptorantibodies, are known in the prior art.

In addition to the specifically mentioned sequences, which code forsingle chain IL-12 and a costimulator protein, the vectors according tothe invention may also contain sequences encoding one or several othercytokines that activate T- and/or B-cells or encoding one or severalother costimulator proteins.

In particular the present invention relates to vectors comprisingsequences coding for single chain IL-12, 4-1BB ligand and IL-2.According to the present invention a protein is referred to as IL-2,when it is coded by a sequence having a sequence homology of at least40%, preferably at least 70%, at least 80% or at least 90% in relationto the sequence presented in FIG. 22. Sequences coding for IL-2 that areused according to the present invention further exhibit essentially theimmune stimulatory activity of the native IL-2, i.e., according to thepresent invention IL-2 coding sequences are used, which exhibit animmune stimulatory activity in vitro corresponding to at least 70% ofthe activity of the native IL-2. Corresponding in vitro assays for thedetection of IL-2 activity are known in the prior art. Preferably, themethod described by Gillis et al. (J. Immunol., 1978, 120 (6): 2027-32)is used to determine activity.

In a further embodiment, the vectors according to the invention inaddition to single chain IL-12, 4-1BB ligand and IL-2 also comprisesequences coding for the costimulator protein B7-1 and/or B7-2.

In the context of the present invention, a costimulator protein isreferred to as B7-1 (or B7-2) if it is coded by a gene with a sequencehomology of at least 40%, preferably of at least 70%, of at least 80% orof at least 90% in relation to the sequences presented in FIG. 23 A (orB).

According to an embodiment of the present invention, the vectors furthercomprise sequences which allow the expression of the coding sequences.The vectors according to the invention may thus contain a promoter andone or more internal ribosome entry sites (IRES). The promoters mayexhibit tumor specificity, that means that they are expressed in thetumor only, or they are not active in all cells.

In certain embodiments of the invention, non-specific promoters arepreferred, because as a rule such promoters are expressed superiorly andsuch vectors may be used for the treatment of different tumors.

According to the invention, particularly high expression rates of theimmune stimulating genes were obtained using vectors which are at leasttri-cistronic and which are further characterized by containing only onepromoter per expression cassette, and by containing one IRES sequencefor each cistron, which is not located directly after a promoter. It hasbeen shown that the use of several promoters within one expressioncassette may lead to cross-inhibitory effects of these promoters. Acombination of promoters and IRES sequences resulted in improvedexpression. The use of different IRES sequences within one vector hasthe further advantage that the recombination frequency between thesesequences may be minimized.

When using tetra-cistronic vectors, it might be advantageous to splitthe cistrons into several expression cassettes (see examples). In thiscase, preferably one promoter is present per expression cassette. Due tothe split into two expression cassettes that preferably have maximumdistance within the vector, the promoters are spatially separated andcross-inhibition is thus reduced.

The sequences according to the invention exhibit the special advantagethat the proteins are particularly well expressed in human cells. Inthis embodiment the advantageous effect of the sequences according tothe invention for immunotherapy is thus also based on the highexpression of the coding sequences.

In the context of the present invention, the vectors preferably consistof DNA or RNA.

In the context of the present invention, a vector is referred to as“viral vector” if it consists of a nucleic acid sequence that comprisessequences of viral origin which permit packaging of the nucleic acid inviral envelopes.

Dependent on the viral origin of the sequences, the vectors may bepresent as adenoviral vectors, adeno-associated vectors, lentiviralvectors, HSV vectors, retroviral vectors, baculoviral vectors orSemliki-Forrest-Virus vectors. The adenoviral vectors may be adenoviralvectors of the first (deletions in the regions E1 and E3 of the AdEasycloning system; e.g., available from QBiogene GmbH, Heidelberg, Germany)or second generation (deletions in E1, E2, E3, E4, etc.) orhelper-dependent adenoviral vectors. Corresponding vectors areextensively known in the art (Nicklin S A, Baker A H, Curr Gene Ther.,2002, 2: 273-93; Mah et al., Clin Pharmacokinet., 2002, 41: 901-11).

An especially preferred embodiment of the present invention relates tothe invention of an adenoviral vector comprising sequences that encodethe single chain IL-12, 4-1BB ligand and IL-2. Adenoviral vectors havethe special advantage that corresponding vectors exist which areapproved for use in human gene therapy. Therefore, the vectors are safefor certain applications (for example, tumor treatment with localadministration). Adenoviral vectors belong to vector systems that areused most frequently in a hospital, and for which most of the dataconcerning safety of their use are available.

However, only a limited amount of foreign nucleic acid sequences can beincorporated in adenoviral vectors. Until now it was therefore notpossible to incorporate more than two genes that encode immunestimulatory proteins into a corresponding vector. This problem is solvedfor the first time by the subsequent detailed description of the vectordevelopment and thus adenoviral vectors are enabled which encode moreimmune stimulatory proteins as compared with known vectors. Particularlyadvantageous adenoviral vectors for 3 genes (3-gene-vectors) are thusprovided by means of the present invention, that may also be referred toas tri-cistronic vectors. Further, for the first time, adenoviralvectors are provided that express 4 genes, wherein in the presentedexamples they were split up into two expression cassettes.

The present invention further relates to virus particles that comprisethe vectors according to the invention. The term virus particles orvirions refers to nucleic acids that are enclosed by the coat proteinsof a virus.

According to a further embodiment, the present invention relates tomedicaments comprising the vectors or virus particles according to theinvention. The vectors or virus particles according to the invention maybe mixed with a carrier compound or they can be administered togetherwith further adjuvants. The vectors or virus particles according to theinvention may be, for example, incorporated into liposomes or intoliposomes with replication-competent adenoviruses (RCAs; see Yoon etal., Curr Cancer Drug Targets, 2001, 1: 85-107), or may be present aspolyethylene glycol enclosed adenoviruses, as antibody-linkedadenoviruses (i.e., as viruses linked with an antibody that hasspecificity for the virus and for a cellular marker, preferably a tumorcell marker), admixed with RCAs, as a cassette within an RCA or as anRCA conditioned for replication within the tumor (conditional RCA: RCAwith an E1 function under the regulation of a tumor specific promoter;van der Poel et al., J Urol., 2002, 168: 266-72).

The exact dosage of the virus particles depends on the disease to betreated, on the type of administration and on the structure of the usedvector, and may be determined by the expert in an individual case usingstandard procedures. The nucleic acids according to the invention enablea destruction or a significant reduction of the tumor already at anespecially low dosage. The medicament preferably has a concentration perunit dose of not more that 1×10¹¹, preferably of not more than 1×10¹⁰,not more than 1×10⁹ or not more than 1×10⁷. However, the dosage may besignificantly lower than the mentioned ranges and may even be as low as1×10⁶. The dosage numbers given here refer to the number of infectiousvirus particles. It has been shown in a rat tumor model that all animalssurvived tumor injection over a period of more than one year whentreated with a dosage of 5×10⁷ infectious virus particles. Stillapproximately 90% of the animals survived a treatment with a dosage of5×10⁶ infectious virus particles. However, all animals in the controlgroup died within the first 50 days after the tumor injection. Thesedosages are several orders of magnitude below the doses suggested in theprior art for corresponding treatments.

The medicament is formulated such that the vectors are transferredsufficiently to the tumor. Preferably, the medicament is used as asolution for the intra-tumoral injection. The production ofcorresponding solutions is well known in the art. As an alternative, themedicament can be formulated as a carrier compound that releases thevector over a certain time period after implantation into the tumor.Corresponding carrier compounds, e.g., cellulose sulfate or the like,are well known in the art.

Finally, the present invention relates to the use of the vectors or thevirus particles for the treatment of tumors, especially for thetreatment of solid tumors, like HCC, colon cancer, breast cancer, etc.,in humans.

According to an alternative embodiment, the present invention relates tothe use of the vectors or virus particles for the treatment ofinfectious diseases or prionic diseases. An immune stimulating therapy,also in the form of a gene therapy, was already suggested for thetreatment of corresponding infectious diseases (see van der Meide etal., Vaccine., 2002, 20: 2296-302).

The immune stimulating effect of the vectors and virus particlesaccording to the invention thus has further therapeutic potential forthe treatment of infectious diseases, such as for the treatment ofinfections caused by the human immunodeficiency virus (HIV), byhepatitis virus types A, B, C (HAV, HBV, HCV), by Cytomegalovirus (CMV),and by human papilloma viruses (HPV), which among others, can causecervical carcinoma. The viruses or virus particles may also be usedadvantageously for the therapy of prionic diseases, since unspecificimmune stimulation has already resulted in a successful cure in ananimal model (Sethi et al., Lancet, 2002, 360: 229-30).

For the medical use according to the invention, the vector is present ina concentration of not more than 1×10¹¹, preferably of not more than1×10¹⁰, not more than 1×10⁹ or not more than 1×10⁷. However, the dosagemay be considerably below the mentioned ranges and may be lower than1×10⁶. Again, the dosage numbers given here refer to the number ofinfectious virus particles.

The following examples illustrate the invention. Detailed information onthe production and the use of the vectors mentioned in the examples arefurther presented in the doctoral thesis (dissertation) of ReinhardWaehler having the title “Adenovirale Immuntherapie solider Tumore amHCC-modell der Ratte (Rattus norvegicus, Berkenhout 1769)” of the“Fachbereich Biologie”, Hamburg University.

Examples I. Construction of the Vectors Ad-1, Ad-2, Ad-3 and theirTesting In Vitro and In Vivo

1. Production of the Vectors

At first, the murine cDNAs of scIL-12, 4-1BBL and IL-2 were cloned intothe plasmid pTrident3 (FIG. 1). The result was termed pT3scIL-12[IRES]4-1BBL[IRES]IL-2 (not shown). Beforehand, the 5′-regions ofthe reading frames of the three components and their distances to theIRES elements were modified.

The thus constructed tri-cistronic expression cassette was clonedwithout promoter and without the 3′-non-translated sequences into thepShuttle-CMV plasmid of the AdEasy system (QBiogene GmbH, Heidelberg).The result is the plasmid pShuttle [CMV]scIL12[IRES]4-1BBL[IRES]IL2(FIG. 2). The correctness of the cassette[CMV]scIL12[IRES]4-1BBL[IRES]IL2 of the last mentioned plasmid wascompletely verified by DNA sequencing.

Then, after co-transformation with the plasmid pAdEasy1 into the E. colistrain Bj5183, this construct was recombined to the plasmid pAd-3 (seeFIG. 1) that contains the complete recombinant DNA for Ad-3.

Starting from pShuttle-[CMV]scIL12[IRES]4-1BBL[IRES]IL2, the plasmidsfor Ad-2, pShuttle-[CMV]scIL12[IRES]4-1BBL[IRES]1BBL (see Fig.) and forAd-1, pShuttle-[CMV]scIL12 (see FIG. 1) were cloned, and in an analogousmanner the plasmids pAd-2 and pAd-1 were generated for the production ofpAd-3.

After their release from the plasmid precursors by PacI-digestion theadenoviruses were transfected into HEK293 cells and the resulting viralplaques were isolated propagated (i.e., as virus particles).

Overview of the express cassettes: FIG. 1.

2. Expression Tests

2.1 IL-12

Rat hepatoma cells, McA-RH7777, were infected with the viruses (virusparticles) Ad-1, Ad-2 and Ad-3, and the IL-12 expression was quantifiedin the cell culture supernatant after different time points using IL-12p70-ELISA.

Overview: FIG. 2.

2.2 4-1BBL

Rat hepatoma cells, McA-RH7777, were infected with the viruses (virusparticles) Ad-1, Ad-2 and Ad-3, and the 4-1BBL expression was determinedafter different time points by flow-through cytometry after staining ofthe 4-1BBL with the antibody TKS-1 (BD Pharmingen, Heidelberg). Ad-1 wasused as negative control, since no 4-1BBL expression can be expected.

Overview: FIG. 3.

2.3 IL-2

Rat hepatoma cells, McA-RH7777, were infected with the viruses (virusparticles) Ad-1, Ad-2 and Ad-3, and the IL-2 expression was quantifiedin the cell culture supernatant after different time points using IL-2ELISA. Ad-1 and Ad-2 were used as negative controls, because no IL-2expression can be expected (not shown).

Overview: FIG. 4.

3. Testing In Vivo

3.1 Dose Escalation 1

The vector Ad-3 (FIG. 5 a) was tested in our rat model forhepatocellular carcinoma (HCC). The cell line McA-RH7777 (hepatocellularcarcinoma of the rat), which is syngenic to the “Buffalo rat”, is usedfor subcapsular hepatic transplantation of tumors.

The tumor growth after implantation of 1 million cells is monitored bymagnetic resonance tomography (MRT) in cooperation with Prof. Dr. GerritKrupski-Bardien from the radiological clinic of the UKE before and afterinjection of the virus particles. The development of tumor volumesbetween day 3 and day 12 after virus injection is shown in FIG. 5 b. Thevery clear effect of the vector within this short time period isdose-dependent and the highest dose yields a reduction of tumor volumesto 27% of the size at the time of virus injection. This result isdocumented in FIG. 6 with selected MRT images of individual animals toillustrate the tumor size adequately in relation to the animal size.

Based on these data, the dosage for the long-term treatment of largeranimal groups was determined at 5×10⁷ infectious particles per tumor. Insubsequent studies the effect of the individual vectors (Ad-1 to Ad-3)was now determined at this low dose. As shown in the scheme of FIG. 7,tumors were implanted, then after 14 days 6 animals of the A-groups weretreated with the vector and the parameters of the anti-tumor immuneresponse were analyzed after a further 2 weeks. Ten animals eachconstitute the B-groups (long-term group). Different to the doseescalation study (FIGS. 5 and 6) now two tumors were planted, only oneof which was treated with the virus in order to determine the distaleffects of the immune stimulation. The B-groups were used to analyze thelong-term kinetics of the tumor reduction and the survival rate.According to this scheme the surviving animals received a furtherintrahepatic tumor implantation after three months to test the immunememory in relation to recognition and combat of tumor recurrence. Thesurvival rate is again determined for another three months.

Our data also show containment of tumor growth within the first 14-dayperiod for the chosen dose of 5×10⁷. The B-groups were already monitoredfor 100 days. The results of the tumor reduction by our treatment areillustrated in the form of MRT images in FIGS. 8 to 10.

The figures show that with the chosen dose within 7 weeks after virusadministration a complete removal was achieved for the injected hepatictumors but also for the non-injected tumors. The further effect of acomplete removal (see Ad-2 series in FIGS. 8 to 10) also of metastasesin the liver and in the peritoneum (Ad-2, week 3) is impressive.

The development of the tumor volumes determined from the MRT data isshown and discussed in FIG. 11.

The course of the study expressed as survival rate in % within thetreated groups is illustrated in FIG. 12. In the course of the animalexperiments IL-12 was detected in the serum of the rats. Additionally,interferon-gamma was determined which is released from immune cells andwhich is responsible for the majority of anti-tumor effects.Interferon-gamma was also clearly detectable. In addition to thesedeterminations, T-cells which are specifically directed against thetumor were detected in a so-called cytotoxicity test. The immune cellresponse was actually also characterized with tissue preparations of thetreated animals. In treated tumor tissue CD8+ cells, CD4+ cells,macrophages and natural killer cells were detected in increased amountsas compared to tissue treated with control vector.

3.2 Protocol for Performing the Long-Term Study

Two tumors were planted by injection of McA-RH7777 cells. The cells wereinjected into two different liver lobes. For one of the tumors 1 millioncells were used. In this left-side tumor the virus injection wasperformed later. A further right-side tumor was planted with 650,000cells. This tumor served as an intrahepatic metastasis model. At thistumor the efficacy of the stimulated immune cells in a distal tumor waschecked. The results are shown in FIGS. 9 to 12 and 28 to 30.

II. Construction of Ad-4

Using an insertion system different from homologous recombination in E.coli cells, an expression cassette for the B7-1 is inserted at theposition of the adenoviral E3 region. This region is functionallyinactive, because large segments of this region were deleted in thevectors used here. The expression cassette includes its own promoter ofthe human phosphoglycerate kinase or a promoter with similar strength.

The production of the viruses was performed in an equivalent manner asdescribed in the preceding protocol.

1. An adenoviral vector comprising a first nucleic acid sequence havinga sequence homology of at least 80% to the full length sequence setforth in SEQ ID NO: 2; a second nucleic acid sequence having a sequencehomology of at least 80% to the full length sequence set forth in SEQ IDNO: 3, a third nucleic acid sequence having a sequence homology of atleast 80% to the full length sequence selected from the group consistingof SEQ ID NOs: 4, 6, and 7; and a fourth nucleic acid sequence having asequence homology of at least 80% to the full length sequence set forthin SEQ ID NO: 5; wherein said first nucleic acid sequence and saidsecond nucleic acid sequence are linked together and when expressedproduce a single fusion protein.
 2. The adenoviral vector according toclaim 1, further comprising one or several promoters and one or severalinternal ribosome entry sites (IRES).
 3. The adenoviral vector accordingto claim 1, wherein said adenoviral vector is an adenoviral vector ofthe first or second generation or a helper-dependent adenoviral vector.4. The adenoviral vector according to claim 1, wherein said one orseveral promoters is a non-tumor-specific promoter that mediates theexpression of said first and said second nucleic acid sequences and saidone or several IRES sequences precedes said third nucleic acid sequenceand said fourth nucleic acid sequence.
 5. A virus particle comprisingthe adenoviral vector according to claim
 1. 6. A medicament comprisingvirus particles according to claim
 5. 7. A medicament comprising theadenoviral vector according to claim
 1. 8. A medicament according toclaim 7, wherein said medicament is formulated as a solution forintra-tumoral injection or as a carrier compound that releases theadenoviral vector over a certain time period after implantation into thetumor.
 9. The adenoviral vector according to claim 1, wherein said thirdnucleic acid sequence comprises a nucleic acid sequence having asequence homology of at least 80% to the full length sequence of SEQ IDNO:
 4. 10. An adenoviral vector comprising a first nucleic acid sequencehaving a sequence homology of at least 80% to the full length sequenceset forth in SEQ ID NO: 2; a second nucleic acid sequence having asequence homology of at least 80% to the full length sequence set forthin SEQ ID NO: 3, and a third nucleic acid sequence having a sequencehomology of at least 80% to the full length sequence selected from thegroup consisting of SEQ ID NOs: 4, 6, and 7; wherein said first nucleicacid sequence and said second nucleic acid sequence are linked togetherand when expressed produce a single fusion protein.
 11. The adenoviralvector according to claim 10, further comprising one or severalpromoters and one or several internal ribosome entry sites (IRES). 12.The adenoviral vector according to claim 10, wherein said adenoviralvector is an adenoviral vector of the first or second generation or ahelper-dependent adenoviral vector.
 13. The adenoviral vector accordingto claim 11, wherein said one or several promoters is anon-tumor-specific promoter that mediates the expression of said firstand said second nucleic acid sequences and said one or several IRESsequences precedes said third nucleic acid sequence.
 14. The adenoviralvector according to claim 10, wherein said third nucleic acid sequencecomprises a nucleic sequence having a sequence homology of at least 80%to the full length sequence of SEQ ID NO:
 4. 15. A virus particlecomprising the adenoviral vector according to claim
 10. 16. A medicamentcomprising the adenoviral vector according to claim
 10. 17. A medicamentcomprising virus particles according to claim
 15. 18. A medicamentaccording to claim 16, wherein said medicament is formulated as asolution for intra-tumoral injection or as a carrier compound thatreleases the adenoviral vector over a certain time period afterimplantation into the tumor.